Proteome-wide identification of NEDD8 modification sites reveals distinct proteomes for canonical and atypical NEDDylation

The ubiquitin-like molecule NEDD8 controls several biological processes and is a promising target for therapeutic intervention. NEDDylation occurs through specific NEDD8 enzymes (canonical) or enzymes of the ubiquitin system (atypical). Identification of NEDD8 sites on substrates is critical for delineating the processes controlled by NEDDylation. By combining the use of the NEDD8 R74K mutant with anti-di-glycine (anti-diGly) antibodies, we identified 1,101 unique NEDDylation sites in 620 proteins. Bioinformatics analysis reveals that canonical and atypical NEDDylation have distinct proteomes; the spliceosome/mRNA surveillance/DNA replication and ribosome/proteasome, respectively. The data also reveal the formation of poly-NEDD8, hybrid NEDD8-ubiquitin, and NEDD8-SUMO-2 chains as potential molecular signals. In particular, NEDD8-SUMO-2 chains are induced upon proteotoxic stress (atypical) through NEDDylation of K11 in SUMO-2, and conjugates accumulate in previously described nucleolus-related inclusions. The study uncovers a diverse proteome for NEDDylation and is consistent with the concept of extensive cross-talk between ubiquitin and Ubls under proteotoxic stress conditions. [Display omitted] •A diGly-based approach for the proteome-wide identification of NEDD8 modification sites•Distinct proteomes for canonical and atypical NEDDylation•Existence of diverse poly-NEDD8, hybrid NEDD8-ubiquitin, and NEDD8-SUMO-2 polymers•NEDD8-SUMO-2 chains accumulate in nucleolus-related inclusions upon proteotoxic stress Identification of modification sites for ubiquitin/Ubls is critical for elucidating the regulated processes. Lobato-Gil et al. report the proteome-wide identification of NEDD8 sites, which defines distinct NEDD8 proteomes for canonical and atypical NEDDylation and formation of diverse homo- and hybrid NEDD8 polymers. Proteotoxic-stress-induced NEDD8-SUMO-2 chains accumulate in nucleolus-related inclusions.